孓遗植物桫椤RAPD分析中主要反应参数的影响

The effects of several amplification conditions on RAPD amplification for Alsophila spinulosa

为得到桫椤RAPD扩增的最佳条件,对影响桫椤RAPD扩增的模板纯化方法、模板含量、酶浓度、Mg^(2+)浓度、dNTP浓度和引物浓度及扩增程序等多种因素进行了探讨。结果表明,用玻璃奶和蛋白酶K纯化后获得的DNA作为模板进行RAPD反应,其扩增带比用未纯化的和仅用无水乙醇再沉淀的DNA为模板更亮,更清晰,重复性更好;模板浓度、酶浓度、Mg^(2+)及dNIT浓度、引物浓度及退火温度这些因素都会对RAPD产生影响。经过本实验的探索,发现桫椤RAPD扩增的最佳体系是(2.5×10^(-2)mL反应体积):模板浓度为70ng,Mg^(2+)浓度为2.5mmol·L^(-1),dNTP为0.2mmol·L^(-1),引物3×10^(-4)mL;最佳扩增程序为:94℃200s,一个循环;94℃60s,36℃60s,72℃120s,40个循环:72℃600s,一个循环:4℃保存。

To get the optial amplification conditions for Alsophila spinulosa,the effects of the purified methods of DNA templates and the concentrations of DNA templates,Taq DNA polymerase,magesium,primer and dNTP are tested.The results suggest:the purified templates by glass milk or proteinase K are brighter,clearer and more repeatable than the unpurified templates or precipitated templates by ethanol;the concentration of DNA templates,Taq DNA polymerase,magesium,primer and dNTP will influence the productions of RAPD.The optimal amplification conditions for Alsphila spinulosa is acquired through the experiments:the concentration of DNA templates is 70ng,the concentration of magnesium is 2.5 mmol-L-1,dNTP is 0.2 mmol-L-1,primer is 3 X 10~4 mL.The optical amplification program is also obtained:94 ?00s,1 cycle;94C 60s,36C 60s;72C 120s,40 cycle;72C 600s,1 cycle;stored in 4C.